Dissemination Pattern of Hospital-Acquired Methicillin-Resistant Staphylococcus aureus and Community-Acquired MRSA Isolates from Malaysian Hospitals: A Review from a Molecular Perspective

The global emergence of methicillin-resistant Staphylococcus aureus (MRSA) that unsusceptible to a wide selection of antimicrobial agents and any newly introduced antimicrobial over the past decades has triggered more extensive holistic measures to put an end to this situation. Molecular surveillance of MRSA clones is important to understand their evolutionary dynamics for investigating outbreaks, propagating precautionary measures, as well as planning for appropriate treatment. This review includes peer-reviewed reports on the molecular characterisation of clinical Staphylococcus aureus isolates within Malaysian hospitals from year 2008 to 2020. This work highlights the molecular clones of hospital-acquired MRSA (HA-MRSA) and community-acquired MRSA (CA-MRSA) isolates from Malaysian hospitals, with description on their ever-changing pattern. Among HA-MRSA, the ST22-t032-SCCmec IV MRSA clone was reported to supplant the previous dominating clone, ST239-t037-SCCmec III. Meanwhile, ST30, ST772, ST6 and ST22 were repeatedly detected in CA-MRSA, however, none of the strains became predominant. Future in-depth study on molecular epidemiology of MRSA clone is essential for the investigation of the extent of the clonal shift, especially in Malaysia.


Dissemination Pattern of Hospital-Acquired Methicillin-Resistant Staphylococcus aureus
and Community-Acquired MRSA Isolates from Malaysian Hospitals: A Review from a Molecular Perspective main reason behind Staphylococcus aureus environmental adaptation (4, 6). Examples of such Staphylococcus aureus adaptability are the acquisition of resistance to newly introduced drugs and the continuous emergence of new strains.
One key feature that renders Staphylococcus aureus infection a major concern is its ability to acquire resistance to the newly introduced drug. Formerly, penicillin was the drug of choice to treat Staphylococcus aureus infections. Then, this pathogen developed resistance to penicillin after a year of its usage. Methicillin-resistant Staphylococcus aureus (MRSA) emerged after a year of the introduction of semisynthetic antibiotic methicillin. MRSA is referred to the reduced susceptibility of Staphylococcus aureus to methicillin and other beta-lactam-related drugs by acquiring SCC mec (SCCmec) elements in the bacterial chromosome (7). The SCCmec includes the mecA gene and cassette chromosome recombinases (ccr) gene that encodes methicillin resistance property and recombinases for site-specific integration into the bacterial chromosome. The mecA gene encodes for a variant of penicillin-binding protein 2A (PBP2A) that may resist beta-lactam inhibition of cell wall synthesis (7). MRSA first emerged in the 1960s in Asian countries (8), whereas MRSA isolates were first reported in 1971 in Malaysia at a university hospital (9). Based on the mode of acquisition, MRSA can be classified as hospital-acquired MRSA (HA-MRSA) and community-acquired MRSA (CA-MRSA).
Another example of Staphylococcus aureus adaptability is the blurred line between the distinctive genetic characteristics of HA-MRSA and CA-MRSA. The ST22-SCCmec IV strain was originally considered a CA-MRSA strain at its early emergence and has long become an epidemic in hospital settings worldwide. In the early days of its discovery, CA-MRSA was usually associated with the PVL gene rather than HA-MRSA (10). Due to the distinctive genetic characteristics of the MRSA isolates based on the PVL status and the evidence of leucocidin causing increased virulence in the Staphylococcus aureus strain, the PVL gene was typically incorporated in the molecular typing of MRSA isolates (11). However, the PVL gene has lost its exclusivity to CA-MRSA upon reports of the latter harbouring the gene (12). This dynamic clonal change of MRSA strains could also change the pathogenesis of the infections and their Introduction Staphylococcus aureus is a Gram-positive bacterium known both as normal flora and a human pathogen that causes mild to lifethreatening infections, such as skin and soft tissue infection (SSTI), food poisoning, pneumonia, infective endocarditis, osteomyelitis and bacteraemia (1). This bacterium possesses several exoproteins and toxins to adapt to a host and induce diseases. One of the important toxins is Panton-Valentine leukocidin (PVL). The PVL is a toxin composed of bicomponents, LukS-PV and LukF-PV, encoded by lukS-PV and lukF-PV genes, respectively. Both of these two components are secreted before they are assembled into a pore-forming heptamer on neutrophil membranes, leading to neutrophil lysis. Normally, this toxin is linked to skin and soft tissue infection, communityacquired infection, recurrent infection without predisposing factors, and severe infections such as necrotising pneumonia and severe musculoskeletal infection (2, 3). Other virulence factors include staphylococcal enterotoxin A to E (encoded by sea, seb, sec, sed and see genes), protein A (spa gene), exfoliative toxin (eta and etb genes), alpha-haemolysin and deltahaemolysin (hla and hld genes) and toxic shock syndrome toxin 1 (tst gene) (4). The virulence factors contribute to the pathogenicity of staphylococcal foodborne disease and toxic shock syndrome, pathogen binding to Fc domain of host immunoglobulin G, association with staphylococcal scalded skin syndrome (SSSS), cytolytic toxin particularly to human platelets and monocytes, cytolytic activity with high affinity to leucocytes, thereby causing toxic shock syndrome (TSS) and SSTI (4).
Apart from the abovementioned virulent determinant genes, Staphylococcus aureus genetic components also comprise antibiotic resistance genes that are mostly encoded on mobile genetic elements (MGEs) (5). Normally, they are defined as DNA fragments that encode a range of virulence and resistance factors, along with enzymes that initiate their transition and incorporation into new host DNA. The MGEs in Staphylococcus aureus consist of staphylococcal cassette chromosome (SCC), transposons, integrative conjugative elements (ICEs), integrons, bacteriophages, pathogenicity islands and plasmids (4, 6). These MGEs are movable among bacteria through horizontal gene transfer (HGT) and are believed to be the typing were chosen. For this review, a total of 18 relevant articles were identified and listed in Table 1.

Clonal Pattern of Hospital Acquired MRSA Clones in Malaysian Hospitals
A total of five (types I, II, III, IV and V) out of 13 SCCmec types of MRSA were reported in Malaysia (Table 1) (31). The study reported that one of the type II isolates was associated with ST239. The same study also claimed that no ST239-II clone was reported in previous studies, including in Japan and Korea where SCCmec type II is common. However, a study involving several Korean hospitals MRSA isolates in 2003 and 2004 reported circulation of this clone (34). This clone has been associated with numerous hospital transmissions (35), but this case has yet to be investigated in Malaysia. This could be due to the insignificant number of type II found in the country (10, 21). Interestingly, type II, typical of HA-MRSA strain, was reported to be isolated among CA-MRSA in a study in West Malaysia (36), demonstrating the establishment of HA-MRSA strain in the community setting.
Most studies involving Malaysian clinical isolates inferred that ST239-SCCmec type III is the most circulating clone in the hospital setting (10, 24, 29, 30, 33, 37, 38). ST239-III is a singlelocus variant of ST8 found to be the predominant clone in most Asian countries, including from neighbouring countries such as Indonesia, Lao People's Democratic Republic, Vietnam and Thailand (39)(40)(41)(42)(43). ST239-III, also known as the Brazilian/Hungarian epidemic clone, was first detected in Brazil in 1995 and later identified in Hungary in 1997, as the name perceived. Between 1998 and 2003, the ST239 strain disappeared from the Hungarian hospitals and response to treatments. Hence, it is critical to study the dynamic changing epidemiology of MRSA to evaluate the effectiveness of the existing control measures, gain insight into its evolutionary mechanism and limit its spread.
Further to the above, several MRSA surveillance networks have been established worldwide to control the dissemination of MRSA based on the antibiotic resistance profile and molecular background (13-18). The World Health Organization (WHO) initiated the Global Antimicrobial Resistance Surveillance System (GLASS) in 2014 (19) with the aim to harmonise the established surveillance systems, particularly on antibiotic resistance organisms, and undoubtedly, the antibiotic resistance data from Malaysian hospitals as well. Similarly, Malaysian MRSA antibiotic resistance prevalence is accumulated and reported nationally via the Malaysian National Surveillance on Antimicrobial Resistance (NSAR) (20). In Malaysia, molecular-based surveillance is maintained at small-scale initiatives by several hospitals and laboratories (21-26).
There are several documented events on the rise, decline and replacement of predominant MRSA strains. For example, in Hungary, ST239-SCCmec III was the most predominant clone during the 1990s. In the early 2000s, the clone disappeared and was replaced by ST228-SCCmec I and epidemic ST5-SCCmec II clones (27). The distribution of MRSA clones in Malaysia has been well explored; however, summary of MRSA clonal changes has never been collectively examined. As such, this review describes the distribution and possible clonal changing pattern of Malaysian hospital on MRSA isolates through a summary of published literature on MRSA clones. This review includes studies that reported the molecular characterisation of clinical Staphylococcus aureus isolates within Malaysian hospitals. Several studies that were published from 2008 until 2020 were searched using the Medline (via PubMed) and Embase databases based on the following terms with the aid of appropriate Boolean operators: methicillin-resistant Staphylococcus aureus, MRSA, molecular, clonal, hospital, clinical and Malaysia. The relevance of the abstract or title of the papers obtained to the review topic was evaluated. Additional publications were culled from the bibliographies. Publication reporting molecular typing of MRSA based on SCCmec typing, multilocus sequence typing (MLST) and spa in a single tertiary hospital. Hence, there is a need for continuous molecular surveillance of MRSA with a wider geographical coverage to establish the possible dissemination of the clone in Malaysia.
Additionally, there were other common HA-MRSA clones reported among HA-MRSA infections, such as ST45-IV (Berlin clone), ST5-t002 (paediatric clone), ST30-IV (Southwest Pacific clone) and USA-400 (ST1-V), originally assigned as CA-MRSA clones. In Vietnam, ST5 and ST45 were reported among the CA-MRSA isolates (48). Fortunately, these clones have never become pandemic in the local settings. Table 1 lists the molecular genotypes of Malaysian HA-MRSA.

Clonal Pattern of Community-Acquired MRSA Clones in Malaysian Hospitals
Based on previous studies, the number of clinical CA-MRSA cases reported in Malaysia varies from 5.6% to 44.4% of the total MRSA clinical isolates from the Malaysian hospitals (8, 12, 15, 32, 49). The SCCmec type IV preponderated Malaysian clinical CA-MRSA isolates, except in the studies by Sit et al. (10) and Neela et al. (29) where they reported type V and type III as the most common strains of CA-MRSA clinical isolates.
ST30-SCCmec type IV, also called the South-West Pacific clone, is the most predominant clonal type of clinical CA-MRSA. A study by Amit et al. (36) reported spa-type t019, which is associated with ST30, in their CA-MRSA infection isolates. ST30-IV-spa t019 is a pandemic clone and has been documented to replace the predominant CA-MRSA clones in Argentina, with more aggressive behaviour and higher invasion capacity (50). Fortunately, the distribution of ST30 in Malaysia was sporadic and not persistently detected. No ST30 was isolated from the latest CA-MRSA clinical isolates (23).
Over the past 15 years, ST772-SCCmec type V has been persistently observed albeit at low rates in several local clinical studies involving CA-MRSA (Table 1). This multidrugresistant clone first emerged in Bangladesh and India with the name of Bengal Bay clone, which then disseminated globally, triggering a minor outbreak. Intercontinental clone transmission is typically associated with travel or family contact in Bangladesh and India (47,51). Moreover, sporadic transmission of ST772-V has been reported in several countries, such as was substantially succeeded by ST228-SCCmec type I and ST5-SCCmec type II (27).
Most ST239-III isolates in Malaysia are spa-type t037 (25, 29). Interestingly, a study by Lim et al. (33) showed that the spa-types among ST239 isolates illustrated diversification with an increase in the year of sampling. While the diversity trend has not been statistically confirmed and emphasised in the study, continuous clone surveillance should be performed to elucidate the diversification of MRSA clones in the country. Since 2007, the variability of ST239 in Singapore was again observed following the occurrence of ST22 supplanting ST239 in the early 2000s (44). Moreover, increased diversity of MRSA clones was observed in countries with low MRSA incidence (45). The ease of travelling may aid in the dissemination of the clone into multiple geographical areas, besides the intense selective pressure of the clone itself through mutation in the capsule gene, acquisition of the resistance gene, acquisition of the toxin gene, acquisition of the mobile genetic element and host population factors (46,47).
Apart from ST239, ST22-IV was also described among the isolates since 2003. Subsequently, detection of ST22 remained in low incidence except in two different studies in 2008 and 2010 (25, 31) where no ST22-IV was isolated. ST22-IV, or epidemic MRSA-15 (EMRSA-15), is a dominant clone regularly found in the UK. This clone can spread and establish itself as the most circulating clone, where it displaced the Iberian and Brazilian clones, the clones commonly found among MRSA isolates in Europe. The clone is particularly interesting because it was also the case in the neighbouring country, Singapore, where ST22 superseded ST239 as the standard sequence type in the country. This phenomenon also occurred among the Malaysian clinical isolates. In a study involving MRSA clinical isolates in 2014 and 2015, ST22-IV was the most prevalent clone identified instead of ST239-III (23). However, a more recent study found that SCCmec IV and t032 predominated the MRSA isolates in 2017 (24). Based on the spa-typing database (https:// www.spaserver.ridom.de/), the MLST of the t032 is found to be ST22. Hence, this further supports the replacement of MRSA clones. The smaller SCCmec type IV elements and the specific virulence factors harboured by this clone promote pathogenicity and persistence. However, such a molecular trend was reported ..        Reports on molecular epidemiology of MRSA isolates in Malaysian hospitals have indicated infiltration of the HA-MRSA strain into the community and vice versa, and the PVL gene is increasingly becoming non-exclusive to CA-MRSA isolates. Due to these reasons, comprehensive genotyping for epidemiology and revolutionary study is essential in tracking potential outbreaks, assessing currently adopted precautionary measures and preparing for prophylaxis. In Malaysia, MRSA resistance data is reported consistently through GLASS, NSAR, and other small-scale molecular surveillance by several institutions. Nevertheless, such surveillance efforts are not consistent and not widespread across the country, especially in East Malaysia. A systematic and consistent molecular surveillance of MRSA has proven to lower the dissemination of the pathogen in the European countries (56). Hence, adopting a similar effort could aid in curbing MRSA spread in Malaysia. Currently, researchers worldwide have adopted an in-depth understanding of MRSA epidemiology, innovation and genetic content enabled by whole genome sequencing (WGS). However, such comprehensive use of WGS has never been recorded in Malaysia, as little-to-no local MRSA genome comparison studies have been performed. This could be attributable to the limited access to WGS and the cost of running large-scale WGS beyond the scope of many reference laboratories in Malaysia, especially for genomic data interpretation. With the potential availability of simplified hardware and software packages for genome data generation and analysis, the incorporation of genome analysis in future research and clinical diagnostic use will hopefully be realised in the near future.

CA
Taiwan, Japan, China, Singapore and Myanmar (51, 52) but its persistence among Malaysian isolates should have raised the alarm, as this clone was previously reported causing hospital and household outbreaks in Norway, where MRSA incidence is low (51). A similar situation occurs in Cambodia, where ST834 and ST121 are considered persistent clones among their children CA-MRSA isolates (53). The two STs were first reported in 2009 and consistently detected in the same population in 2011. In 2011, the ST834 strain was also associated with nosocomial infection, indicating that the initially classified as CA-MRSA strain can adapt in hospital settings.
Other clones associated with community characteristics in the hospital infections are ST6-IV, ST1-V, ST80-IV, ST5-V, ST508, ST239-III and ST22-IV (Table 1). In contrast to HA-MRSA-related epidemic transmission, CA-MRSA-related transmission is usually sporadic and rarely causes epidemic outside the region of origins, such as ST8 or USA300 and ST80 in the USA and Europe (51). This is also true in Malaysia, where most of the clones are detected sporadically in low counts. The appearance, disappearance and reappearance of some clones are common. For instance, no further detection of ST1-V among local clinical CA-MRSA isolates was documented after it was first isolated by Neela et al. (29). Moreover, documentation of ST6-IV, ST5-V, ST508 and ST80-IV showed similar patterns in Malaysia. The recent emergence of ST239-III and ST22-IV in the community added evidence of infiltration of HA-MRSA clones into the community settings.
Another molecular characteristic linked to CA-MRSA is the presence of the PVL gene, which is also true for MRSA isolates in Malaysia. It was reported that 88% to 100% of CA-MRSA isolates in previous studies were positive for PVL gene (21, 28,29,36,54,55). Nevertheless, there were also reports on PVL negative among CA-MRSA isolates, as listed in Table 1.

In
Malaysia, although HA-MRSA is predominated by ST239-t037-III, its replacement by CA-MRSA clone, ST22-t032-IV, is evident. ST30 is the most circulating clone among the community setting. The ease of travelling causes the spread of some clones from their geographical origin to a much wider